In today's world, cardiovascular diseases are the leading cause of death (29% of all deaths in 2002 (The world health report 2004)): hypertension and stroke (one of its most severe complications), atherosclerosis, ischemic brain disease (which is in most cases caused by atherosclerosis), and finally, ischemic heart disease and acute myocardial infarction (as its manifestation).
In a broad sense, infarction implies necrosis of part of organ due to insufficiency of blood supply, or mechanical or bacterial/viral lesions. Usually, infarction is a consequence of ischemia—the situation in which a lumen-bearing blood vessel becomes narrowed due to atherosclerotic plaque, or a vessel becomes obstructed by a thrombus or compressed by any entity (e.g., cyst or tumor). According to WHO estimates, ischemic heart disease and its consequences resulted in 12.6% of deaths in 2002 (The world health report 2004). In industrialized countries, this proportion is even higher—30%.
Anti-ischemic measures include several interrelated stages: prevention of ischemia or its treatment and prevention of its most severe consequences, such as infarction. The prevention includes reduction of risk factors, primarily atherosclerosis development: smoking cessation, reduction the level of low density lipoproteins (LDL) in the blood, diet, lifestyle, etc. In the development of ischemia, the primary goal is to restore blood supply (reperfusion) of tissue within 1.5-2 hours after onset of ischemia, and for that purpose, vasodilators (e.g., nitroglycerin), anticoagulants (aspirin, heparin), thrombolytic agents, beta-adrenergic blockers relieving stress and reducing oxygen demand in ischemic tissue, oxygen therapy, and finally, surgical methods, such as angioplasty, bypass surgery, etc. are used.
Reperfusion of tissue, especially after long-term ischemia, is accompanied by the accumulation of reactive oxygen species (ROS) (J. L. Zweier, J. T. Flaherty, M. L. Weisfeldt. Direct measurement of free radical generation following reperfusion of ischemic myocardium. 1987, PNAS USA, 84, 1404-1407). During ischemia, the partial pressure of oxygen in cells decreases, electron carriers of the mitochondrial respiratory chain are reduced that leads to enhanced generation of reactive oxygen species (superoxide anion radical at first, then hydrogen peroxide), accompanied by transition of iron atoms from the ferric state (Fe3+) to the ferrous states (Fe2+). This facilitates the Fenton reaction, thus generating a powerful oxidant, the OH* radical. Neutrophils attracted to the ischemic focus also actively release superoxide and hydrogen peroxide on the background of increased oxygen delivery during reperfusion.
All this leads to the activation of free-radical oxidation (A. J. Tompkins, L. S. Burwell, S. B. Digemess, C. Zaragoza, W. L. Holman and P. S. Brookes. Mitochondrial dysfunction in cardiac ischemia-reperfusion injury: ROS from complex I, without inhibition. 2006, Biochim Biophys Acta. 1762, 2, 223-231), and as a consequence, the development of oxidative stress. The latter leads to consequences which are often more severe than ischemic blood circulatory disorders per se (Vanden Hoek T L, Shao Z, Li C, Zak R, Schumacker P T, Becker L B. Reperfusion injury in cardiac myocytes after simulated ischemia. 1996, Am. J. Phys., 270, 1334-1341). Free radicals have a direct damaging effect on intracellular protein structures, nucleic acids, as well as various membranes; peroxidation of polyunsaturated fatty acids embedded in the membranes, in turn, disturbs the barrier properties of the membranes and leads to perturbed ion homeostasis (in the case of reperfused heart muscle, lipid peroxidation and the peroxidation-induced ion imbalance are considered as one of the leading causes of reperfusion cardiac arrhythmias). In addition, free radical compounds initiate vasoconstriction and hypercoagulability, and accelerated degradation of NO which mediates vasorelaxant (in this situation—anti-ischemic) action.
Namely ROS are considered to be one of the key factors triggering the mechanism of necrosis and apoptosis in ischemic tissue. Therefore, preventing the synthesis of mitochondrial ROS or neutralization of the latter is crucial for survival or recovery of the function of ischemic tissue cells.
Attempts to reduce reperfusion-induced oxidative stress in ischemic tissue were made repeatedly. For example, local hypoxia—reduction of perfusate oxygen content entering the ischemic heart during the first few minutes of reperfusion, suppressed ROS generation and had a protective effect on mitochondria (G. Petrosillo, N. Di Venosa, F. M. Ruggiero, M. Pistolese, D. D'Agostino, E. Tiravanti, T. Fiore, G. Paradies. Mitochondrial dysfunction associated with cardiac ischemia/reperfusion can be attenuated by oxygen tension control. Role of oxygen-free radicals and cardiolipin. 2005, Biochimica et Biophysica Acta, 1710, 78-86).
When ischemic tissue is subjected to hypothermic conditions, ROS production is also reduced, the consequences of ischemia are mitigated, thereby increasing time period during which the tissue can remain under ischemic condition without irreversible changes during the subsequent reperfusion (Riess M. L., Camara A. K. S., Kevin L. G., An J., Stowe D. F. Reduced reactive O2 species formation and preserved mitochondrial NADH and [Ca2+] levels during short-term 17° C. ischemia in intact hearts. 2004, Cardiovascular Research, 61, 580-590). A positive clinical effect of hypothermia was also observed (Hypothermia After Cardiac Arrest Study Group. Mild therapeutic hypothermia to improve the neurologic outcome after cardiac arrest, 2002, New Engl. J. Med., 346, 8, 549-56). However, the clinical applicability of the said method—as well as said local hypoxia—is very limited, primarily because of technical difficulties.
Another way to mitigate the damage caused by ischemia and reperfusion is the use of chelating agents binding free ferrous iron; accumulation of Fe2+ ions in ischemic tissue is one of the factors stimulating a surge in ROS synthesis during reperfusion (the Fenton reaction which occurs with hydroxyl radical formation).
It was shown that the use of chelating agents inhibits ROS synthesis during reperfusion (Spencer K T, Lindower P D, Buettner G R, Kerber R E. Transition metal chelators reduce directly measured myocardial free radical production during reperfusion. 1998, J. Cardiovasc Pharmacol, 32, 3, 343-348), reduces infarct size (C. Demougeot, M. Van Hoecke, N. Bertrand, A. Prigent-Tessier, C. Mossiat, A. Beley, and C. Marie. Cytoprotective Efficacy and Mechanisms of the Liposoluble Iron Chelator 2,2_-Dipyridyl in the Rat Photothrombotic Ischemic Stroke Model. 2004, The Journal of Pharmacology and Experimental Therapeutics, 311, 1080-1087). However, it should be noted that the clinical use of chelating agents is limited since they can cause side effects: for example, long-term use of iron ion chelator such as deferoxamine (especially by young people) can lead to stunted growth, speech disorder, hearing loss, disorder of skeletal formation ((Faa G., Crisponi G. Iron chelating agents in clinical practice. 1999, Coordination Chemistry Reviews, 184, 1, 291-310), heart malfunction and hypotension (Kirschner R E, Fantini G A. Role of iron oxygen-derived free radicals in ischemia-reperfusion injury. 1994b J. Am. Coll. Surg., 179, 103-117).
Finally—since the case in point is ROS-induced damage—it would be logical to assume that preparations with antioxidant effect may also reduce the risk of myocardial infarction and to mitigate the severity of other adverse effects in ischemic tissue. Indeed, it was shown (Kutala V K, Khan M, Mandal R., Potaraju V., Colantuono G., Kumbala D, Kuppusamy P. Prevention of Postischemic Myocardial Reperfusion Injury by the Combined Treatment of NCX-4016 and Tempol. 2006, J Cardiovasc Pharmacol., 48, 3, 79-87), that pre-perfusion of rat heart with antioxidant Tempol reduced infarct size caused by subsequent ischemia/reperfusion by 1.5 times, and the combination of Tempol and NCX-4016 (NO donor)—by almost 2 times. Reduction of ROS production and protection of membrane lipids in ischemic heart mitochondria from peroxidation by means of antioxidant melatonin was demonstrated by Petrosillo et al. (Petrosillo G, Di Venosa N, Pistolese M, Casanova G, Tiravanti E, Colantuono G, Federici A, Paradies G, Ruggiero F. M. Protective effect of melatonin against mitochondrial dysfunction associated with cardiac ischemiareperfusion: role of cardiolipin. 2006, The FASEB Journal, 20, 269-276).
At the same time, attempts to achieve the effect of preventing the development of ischemic processes by means of specialized vitamins-antioxidants (vitamins C, E and beta-carotene), failed to reveal clinical relevance of such prevention (Collins R, Armitage J, Parish S, Sleight P, Peto R; Heart Protection Study Collaborative Group. Effects of cholesterol-lowering with simvastatin on stroke and other major vascular events in 20536 people with cerebrovascular disease or other high-risk conditions. 2004, Lancet, 363, 9411, 757-767). In a review (Kromhout D. Diet and cardiovascular diseases. 2001, J. Nutr. Health Aging, 5, 144-149) on the same issue, similar conclusions were made: convincing clinical evidence for the ability of antioxidants, vitamins E and C as well as carotenoides, to prevent the development of ischemic heart disease was not obtained.
The apparent contradiction between the encouraging results of the experiments on cell cultures or isolated organs, on the one hand, and the disappointing clinical trial data—on the other hand, may be partly explained by “the problem of delivery”. Antioxidant therapy should be conducted at the beginning of reperfusion, the start time of which, in turn, is critical to prevent or minimize the development of myocardial infarction. Antioxidant must not only be promptly delivered to the ischemic region—its intracellular localization is also important. It is possible that namely the inability of existing antioxidant preparations to be quickly and selectively transported into mitochondria, the ROS generation site, is the cause of the low efficiency of such preparations in clinical practice (Becker L. B. New concepts in reactive oxygen species and cardiovascular reperfusion physiology. 2004, Cardiovascular Research, 61, 461-470).
For delivery of antioxidants to mitochondria, a strategy with two different mechanisms of its implementation can be applied. The strategy is based on transport of a required antioxidant to mitochondria which is conjugated with elements transported to the mitochondrial matrix. The nature of these elements is twofold—these can be either penetrating lipophilic cations which can be spontaneously transported along the electric field created on the inner mitochondrial membrane, or signal peptide sequences being part of the transported peptides, after their processing the mature protein is brought into the correct mitochondrial compartment.
To date, very limited number of biologically active compounds is known which can be targeted to mitochondria at the expense of energy of electrochemical potential of hydrogen ions. Among these are mitochondria-targeted antioxidant MitoQ and its variants (MitoQ5, MitoQ3). The said substances are ubiquinol (the reduced form of ubiquinone) attached to triphenylphosphonium through C-10 linker group (C-5, C-3, respectively). In description of the invention U.S. Pat. No. 6,331,532, MitoQ is claimed to be an active compound of compositions intended for the treatment and prevention of diseases associated with oxidative stress. Another compound, mitochondria-targeted mimetic of glutathione peroxidase ebselen is claimed in the claims of invention U.S. Pat. No. 7,109,189 as treatment for adverse effects of reperfusion of ischemic tissue, infarction and as preparation applicable in surgery and transplantation. However, the authors of said invention did not provide any experimental data supporting the possibility of such use of ebselen, since data on the antioxidant properties of ebselen were obtained in experiments on isolated mitochondria and mitochondrial membranes.
Adlam et al. shows an example of the effect of MitoQ observed in an experiment where rats were fed the said compound for 14 days, with subsequent study of the properties of their isolated hearts perfused by Langendorfs method and exposed to ischemia/reperfusion. Said data indirectly confirm the statement that MitoQ can be used for prevention or treatment of ischemic myocardial damage. However, normothermal ischemia followed by reperfusion used by the authors of the said work has all the limitations of in vitro model. Also, given the apparent lack of reliable statistics to assess the number of experiments (6 animals in each group), it remains unclear how much do the average values obtained characterize the changes in the restoration of cardiac function associated with the chemical structure of antioxidants, rather than differences in the development of arrhythmias.
Summarizing this section, one can conclude that at present there is no clinically applicable and experimentally confirmed scheme of treatment and prevention of heart diseases by means of mitochondria-targeted antioxidants.